A large fraction of bacterial signaling is mediated by "two-component" systems that utilize a conserved mechanism of phosphotransfer between a sensor histidine protein kinase and a response regulator protein. Numerous two-component systems are present in a single bacterium and often function in pathways that are important or essential for pathogenesis. The absence of two-component proteins from animals has made them attractive targets for pursuit of new antimicrobial agents. The conserved domain of response regulators functions as a phosphorylation-activated switch, controlling the activity of the effector domain and the output response. This application focuses on structural and biochemical characterization of the mechanism of activation of response regulator proteins. In particular, the project seeks to determine the relative regulatory contributions of intramolecular interactions between the regulatory and effector domains versus intermolecular interactions between the regulatory domains within functional dimers of the large OmpR/PhoB subfamily of response regulators that function as transcription factors. The studies will involve construction and characterization of chimeric response regulator proteins, determination of surfaces of interaction within active dimers, and structural characterization by X-ray crystallography. The applicant's studies are aimed at providing a detailed description of the regulatory mechanisms of a small number of representative response regulators, with the expectation of significance not only to an understanding of these individual proteins, but also to establishing the similarities and differences within a large family of signaling proteins. This system provides an opportunity to address the extent to which sequence and structural similarity can be used to predict similarity in mechanism of function, an important question that is emerging in this era of proteomics. Additional studies are focused on components of bacterial chemotaxis systems that contribute to receptor-mediated adaptation, specifically studies of regulatory interactions in the multi-domain response regulator methylesterase CheB and structural characterization of proteins CheD and CheC.